Ion torrent s5 platform

The V4-V5 variable region of the 16S rRNA gene was amplified using universal primers 515F (5’-GTGCCAGCMGCCGCGG-3’) and 907-R (5’- CCGTCAATTCMTTTRAGTTT-3’). High-throughput sequencing was performed using the Ion Torrent S5 platform at Zhejiang University, China. Operational Units (OTUs) were clustered with a 97% similarity cutoff using USEARCH UPARSE, and the chimeric sequences were removed in the UPARSE pipeline. The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed by USEARCH SINTAX algorithm against the RDP training set (version 18) 16S rRNA database using a confidence threshold of 0.8.
The alpha diversity indices (Chao1, richness, Simpson and Shannon index) and beta-diversity index (Bray-Curtis, Unifrac, Jacaard, and Unifrac-binary distance) were calculated using USEARCH alpha_div and beta_div, respectively. Principal coordinate analysis (PCoA) based on UniFrac-binary distance matrix was plotted by Vegan 2.4.2 and the p value was obtained by permutational multivariate analysis of variance (PERMANOVA). The raw reads count tables at different taxonomic levels (phylum, order, class, family, and genus) were used for differential abundance analysis with DEseq2. Spearman’s rank correlation coefficient was performed to reveal the correlations with the metabolites and the top 50 genera.

Phage DNA was extracted according to Zhao et al. [20 (link),21 (link)] with some modifications. Briefly, the phage was precipitated overnight with 15% (w/v) PEG 8000 and 0.5 M NaCl at 4 °C. Subsequently, the phage was centrifuged at 12,000× g for 20 min and then resuspended in SM buffer. The concentrated phage particles were treated with DNase I (final concentration 0.1 units/μL) and RNase A (final concentration 3 μg/mL) to remove bacterial genomic contamination. Thereafter, the sample was treated with SDS, EDTA, and proteinase K. Finally, the phage DNA was extracted using phenol-chloroform-isoamyl alcohol (25:24:1). The DNA was sequenced using the Ion Torrent S5 platform (Thermo Fisher Scientific, Waltham, MA, USA) and high-quality reads were subsequently assembled using SPAdes v. 3.12.0.